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1.
Nature ; 592(7852): 116-121, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33106671

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.


Assuntos
COVID-19/transmissão , COVID-19/virologia , Aptidão Genética , Mutação , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Cricetinae , Modelos Animais de Doenças , Humanos , Pulmão/virologia , Masculino , Mesocricetus/virologia , Modelos Biológicos , Mucosa Nasal/virologia , Testes de Neutralização , Estabilidade Proteica , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Técnicas de Cultura de Tecidos , Traqueia/virologia , Carga Viral , Vírion/química , Vírion/patogenicidade , Vírion/fisiologia , Replicação Viral/genética
2.
Nature ; 591(7849): 293-299, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33494095

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.


Assuntos
COVID-19/virologia , Furina/metabolismo , Mutação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Pneumopatias/virologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteólise , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genética
3.
J Virol ; 97(2): e0153222, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36722972

RESUMO

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Peptídeos e Proteínas de Sinalização Intracelular , SARS-CoV-2 , Proteínas não Estruturais Virais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/virologia , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/metabolismo , Animais , Cricetinae
4.
PLoS Pathog ; 18(6): e1010627, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35728038

RESUMO

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/genética , Quinase 3 da Glicogênio Sintase , Humanos , Mutação , Nucleocapsídeo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
5.
PLoS Pathog ; 18(5): e1010532, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35533195

RESUMO

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.


Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Ebolavirus/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Transcrição Viral
6.
Proc Natl Acad Sci U S A ; 118(14)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33782133

RESUMO

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Febre do Vale do Rift/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Vero , Proteínas Virais de Fusão/química
8.
PLoS Pathog ; 15(4): e1007610, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947291

RESUMO

The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.


Assuntos
Culicidae/virologia , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Mutação , RNA Polimerase Dependente de RNA/genética , Vertebrados/virologia , Replicação Viral/genética , Animais , Culicidae/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Fenótipo , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Vertebrados/genética
9.
Bioorg Med Chem Lett ; 41: 127983, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33965007

RESUMO

We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.


Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Descoberta de Drogas , Compostos Heterocíclicos/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Arenavirus/metabolismo , Relação Dose-Resposta a Droga , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo
10.
J Virol ; 93(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31462558

RESUMO

Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection.IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.


Assuntos
Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Peptidoglicano/metabolismo , Vírus de RNA/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Flaviviridae/efeitos dos fármacos , Lipopeptídeos/imunologia , Lipopeptídeos/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/metabolismo , Peptidoglicano/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Células Vero , Viroses/metabolismo
11.
Bioorg Med Chem Lett ; 29(22): 126620, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31537423

RESUMO

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.


Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Arenavirus/metabolismo , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo
12.
J Infect Dis ; 218(suppl_5): S438-S447, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30192975

RESUMO

Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.


Assuntos
Doença do Vírus de Marburg/etiologia , Fator de Transcrição STAT2/fisiologia , Animais , Cricetinae , Citocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/patologia
13.
J Cell Sci ; 125(Pt 19): 4576-85, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22797925

RESUMO

FAT10 is a ubiquitin-like modifier proposed to function in apoptosis induction, cell cycle control and NF-κB activation. Upon induction by pro-inflammatory cytokines, hundreds of endogenous substrates become covalently conjugated to FAT10 leading to their proteasomal degradation. Nevertheless, only three substrates have been identified so far to which FAT10 becomes covalently attached through a non-reducible isopeptide bond, and these are the FAT10-conjugating enzyme USE1 which auto-FAT10ylates itself in cis, the tumor suppressor p53 and the ubiquitin-activating enzyme UBE1 (UBA1). To identify additional FAT10 substrates and interaction partners, we used a new monoclonal FAT10-specific antibody to immunopurify endogenous FAT10 conjugates from interferon (IFN)γ-and tumor necrosis factor (TNF)α-stimulated cells for identification by mass spectrometry. In addition to two already known FAT10-interacting proteins, histone deacetylase 6 and UBA6, we identified 569 novel FAT10-interacting proteins involved in different functional pathways such as autophagy, cell cycle regulation, apoptosis and cancer. Thirty-one percent of all identified proteins were categorized as putative covalently linked substrates. One of the identified proteins, the autophagosomal receptor p62/SQSTM1, was further investigated. p62 becomes covalently mono-FAT10ylated at several lysines, and FAT10 colocalizes with p62 in p62 bodies. Strikingly, FAT10ylation of p62 leads to its proteasomal degradation, and prolonged induction of endogenous FAT10 expression by pro-inflammatory cytokines leads to a decrease of endogenous p62. The elucidation of the FAT10 degradome should enable a better understanding of why FAT10 has evolved as an additional transferable tag for proteasomal degradation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteômica , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Autofagia/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Interferon gama/farmacologia , Lisina/metabolismo , Espectrometria de Massas , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteína Sequestossoma-1 , Especificidade por Substrato/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
14.
J Virol ; 87(7): 3710-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23325696

RESUMO

Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.


Assuntos
Proteólise , Vírus da Febre do Flebótomo Napolitano/genética , Proteínas não Estruturais Virais/metabolismo , eIF-2 Quinase/metabolismo , Animais , Western Blotting , Chlorocebus aethiops , Química Click , Primers do DNA/genética , Citometria de Fluxo , Imunofluorescência , Imunoprecipitação , Plasmídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Especificidade da Espécie , Células Vero
15.
Viruses ; 16(7)2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-39066263

RESUMO

Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.


Assuntos
Amidas , Antivirais , Modelos Animais de Doenças , Ebolavirus , Doença pelo Vírus Ebola , Camundongos Endogâmicos BALB C , Pirazinas , Animais , Amidas/uso terapêutico , Amidas/administração & dosagem , Amidas/farmacologia , Cobaias , Pirazinas/administração & dosagem , Pirazinas/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Camundongos , Ebolavirus/efeitos dos fármacos , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Feminino , Vias de Administração de Medicamentos , Esquema de Medicação
16.
J Gen Virol ; 94(Pt 7): 1441-1450, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23515022

RESUMO

Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-ß gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.


Assuntos
Encéfalo/virologia , Doenças do Sistema Nervoso/virologia , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Flebótomo Napolitano/genética , Vírus da Febre do Flebótomo Napolitano/patogenicidade , Vacinas Atenuadas/efeitos adversos , Proteínas não Estruturais Virais/metabolismo , Vacinas Virais/efeitos adversos , Animais , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vírus da Febre do Flebótomo Napolitano/imunologia , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
17.
J Virol ; 86(14): 7650-61, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22573861

RESUMO

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.


Assuntos
Vírus da Febre do Vale do Rift/imunologia , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/imunologia , eIF-2 Quinase/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Quimiocinas/sangue , Chlorocebus aethiops , Cricetinae , Células Dendríticas/imunologia , Interferon-alfa/sangue , Interferon gama/sangue , Interleucina-17/sangue , Interleucina-5/sangue , Camundongos , Mutação , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/patogenicidade , Células Vero , eIF-2 Quinase/antagonistas & inibidores
18.
NPJ Vaccines ; 8(1): 160, 2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37863935

RESUMO

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

19.
bioRxiv ; 2023 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-37131704

RESUMO

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

20.
Emerg Microbes Infect ; 12(1): 2209208, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37114433

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve after its emergence. Given its importance in viral infection and vaccine development, mutations in the viral Spike gene have been studied extensively; however, the impact of mutations outside the Spike gene are poorly understood. Here, we report that a triple deletion (ΔSGF or ΔLSG) in nonstructural protein 6 (nsp6) independently acquired in Alpha and Omicron sublineages of SARS-CoV-2 augments nsp6-mediated antagonism of type-I interferon (IFN-I) signaling. Specifically, these triple deletions enhance the ability of mutant nsp6 to suppress phosphorylation of STAT1 and STAT2. A parental SARS-CoV-2 USA-WA1/2020 strain containing the nsp6 ΔSGF deletion (ΔSGF-WA1) shows reduced susceptibility to IFN-I treatment in vitro, outcompetes the parental strain in human primary airway cultures, and increases virulence in mice; however, the ΔSGF-WA1 virus is less virulent than the Alpha variant (which has the nsp6 ΔSGF deletion and additional mutations in other genes). Analyses of host responses from ΔSGF-WA1-infected mice and primary airway cultures reveal activation of pathways indicative of a cytokine storm. These results provide evidence that mutations outside the Spike protein affect virus-host interactions and may alter pathogenesis of SARS-CoV-2 variants in humans.


Assuntos
COVID-19 , Interferon Tipo I , Humanos , Animais , Camundongos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Interferon Tipo I/metabolismo , Mutação , Glicoproteína da Espícula de Coronavírus
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